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Cancer mutation screening: Comparison of high-resolution melt analysis between two platforms
Henry O Ebili1, Mohammad Ilyas2
1Morbid Anatomy and Histopathology Department, Olabisi Onabanjo University, Ago-Iwoye, Nigeria ; University College Hospital, Ibadan, Nigeria.
Ecancermedicalscience
|May 2, 2015
Summary
High-resolution melt analysis (HRMA) using the ABI 7500 demonstrated high precision and accuracy for cancer mutation screening, comparable to the LightScanner. However, the ABI software struggled to differentiate true mutations from artifacts, giving the LightScanner an edge.
Area of Science:
- Molecular Biology
- Genetics
- Oncology
Background:
- High-resolution melt analysis (HRMA) is an emerging, cost-effective technique for post-polymerase chain reaction (PCR) cancer mutation screening.
- While the LightScanner's HRMA capabilities are well-documented, the performance of the ABI 7500 HRM for this application remains largely uncharacterized.
Purpose of the Study:
- To evaluate the performance of the ABI 7500 HRM for cancer mutation screening by comparing it against the purpose-built LightScanner.
- To assess the internal precision, accuracy, sensitivity, and specificity of the ABI 7500 HRM in detecting KRAS and BRAF mutations.
Main Methods:
- DNA from gastric and colorectal cancer tissues and cell lines was amplified for KRAS (exons 2-4) and BRAF (exons 11, 15).
- Amplified products were analyzed using High-Resolution Melt Analysis (HRMA) on both the ABI 7500 and LightScanner instruments.
- Data were analyzed using ABI HRM software v2.0.1 and LightScanner Call-IT 2.5, with statistical comparisons including concordance, kappa statistics, and AUROC curves.
Main Results:
- The ABI 7500 HRMA exhibited high internal precision, excellent concordance, sensitivity, and specificity when compared to the LightScanner.
- The ABI HRM software v2.0.1 demonstrated limitations in distinguishing true mutations from PCR artifact-induced pseudovariations, unlike the LightScanner software.
- Overall performance of the ABI 7500 HRMA was comparable to the LightScanner, with specific advantages for the LightScanner in artifact differentiation.
Conclusions:
- The ABI 7500 HRM is a capable tool for cancer mutation screening, offering performance comparable to the LightScanner.
- The ABI HRM software requires further development to reliably differentiate true mutations from melting artifacts.
- The LightScanner retains an advantage due to its superior ability to resolve melting curve variations and artifacts.
